Embryonic Stem Cells
ES cell lines are cell lines isolated from the inner cell mass (ICM) of blastocyst-stage embryos, which under specific conditions can be maintained in culture for many passages, i.e. replating of cells onto new cell culture dishes at regular time intervals, without loss of their pluripotency. They maintain a normal karyotype and when reintroduced into a host blastocyst they can colonize the germline. Such cell lines may provide an abundance of pluripotent cells that can be transformed in vitro with DNA (see below), and selected for recombination (homologous or non-homologous) of exogenous DNA into chromosomal DNA, allowing stable incorporation of the desired gene. To date, germline transmission, i.e. the transmission of the ES genome to the next generation, has however only been achieved with ES cells of certain mouse strains.
Murine embryonic stem cells were first isolated in 1981. Since then, several ES cell lines have been established and they are now widely and successfully used for the introduction of targeted mutations or other genetic alterations into the mouse genome. Most of the germline-competent mouse ES cell lines that are in current use have been obtained in the 129 strain, and the remainder in a few other inbred strains (C57BL/6 and crosses with C57BL/6). Furthermore, ES cell lines are at best obtained in 30% of explanted blastocysts, even in the 129 strain, and success rates of around 10% appear to be closer to the norm.
The most commonly used approach to generate chimeric animals is to inject about 10-15 isolated ES cells into the blastocoel of a host blastocyst and to allow the cells to mix with the cells of the inner cell mass. The resultant chimeric blastocysts are then transferred to recipients for rearing. Alternatively diploid aggregation using very early (8-16 cell) stage embryos and tetraploid aggregation, can be used as hosts for ES cells. Briefly, ES cells are ‘sandwiched’ between early stage embryos devoid of their zona pellucida, cultured overnight and implanted into a foster mother. This technique can be performed under conditions yielding either chimeric or totally ES cell-derived offspring.
Although ES cell culture and chimera production have been technically improved over the years, the pluripotency of the ES cells is still often reduced after several passages, whereas completely ES cell-derived fetuses (by tetraploid aggregation) seem to have a markedly reduced survival after birth. Nagy et al., “Derivation of completely cell culture derived mice from early-passage embryonic stem cells” Proc Natl Acad Sci USA 1993; 90: 8424-8, used R1 ES cell lines derived from early passages with electrofusion derived tetraploid embryos to form aggregates and obtained mice which were entirely derived from ES cells. However, the R1 ES cells lost their totipotency upon extended culture in vitro, because no animal survived to adulthood from ES cells obtained from later than 14 passages. Moreover, even when early passage cells were used, many ES-tetraploid aggregates died before developing to term. Only 3.8% of transferred aggregates survived after caesarian section. The goal to obtain viable ES mice using later passage ES cells was not reached and the production of ES cell derived mice using genetically modified ES cells did not seem to be possible.
The inability of the present technology to yield viable offspring from ES cells of inbred mouse strains via tetraploid aggregation was recently confirmed in Eggan K, et al. “Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci 2001; 98: 6209-14. Genetic heterozygosity was found to be a crucial parameter influencing postnatal survival of offspring derived from ES cells by nuclear cloning or tetraploid embryo complementation. Pups derived from inbred ES cells by either method died perinatally with a phenotype of respiratory failure. In contrast, the great majority (80-85%) of pups derived from F1 ES cells by either procedure survived to adulthood. In another study however, no clear correlation was found between the postnatal lethality of ES-cell-derived mice and the cell line used. Postnatal death occurred in all cell lines, including those with different genetic background. Thirty four completely ES-cell-derived newborns (3%) were obtained after transfer of 1037 tetraploid blastocysts injected with ES from all cell lines. Only thirteen mice (1%) grew to adulthood (Amano T, Kato Y, Tsunoda Y. Comparison of heat-treated and tetraploid blastocysts for the production of completely ES-cell-derived mice. Zygote 2001; 9: 153-7).
Presumptive pluripotential ES cells have been isolated from a number of other species than mice, including hamster, pig, sheep, cattle, mink, rat, primate, human, chicken, marmoset, medakafish and man. In only a few instances (pig, chicken, medakafish), have these cell lines given rise to chimeras when reintroduced into blastocysts, but thus far none have given rise to germline transmission.
The isolation of pluripotential ES cell lines from preimplantation rabbit blastocysts was reported by Graves K H, Moreadith R W, “Derivation and characterization of putative pluripotential embryonic stem cells from preimplantation rabbit embryos”, Mol Reprod Dev 1993; 36: 424-33. These ES lines were found to give rise to differentiated cell types, representative of all three germ layers (pluripotential by in vitro criteria). Recently these ES lines from the Dutch Belted strain were shown to be also capable of generating overt coat-color chimeras following injection into recipient New Zealand White blastocysts, demonstrating that the cells were pluripotential by in vivo criteria as well. However no germline transmission has been achieved. Additional experiments showed that the low frequency of chimera formation and absence of germline transmission probably was due to the loss of pluripotency of the ES cell line upon high passage number.
ES cells are maintained in an undifferentiated state by the presence of feeder layers producing various factor(s) that prevent the cells from differentiating. It has been shown that several cytokines are responsible for this effect: DIA/LIF (differentiation inhibitory activity/leukaemia inhibiting factor), interleukin-6 in combination with soluble interleukin-6 receptor, interleukin-11, oncostatin M, ciliary neurotrophic factor and cardiotrophin. It is now possible to establish and maintain ES cells in culture in the absence of feeder cells but in the presence of such factors, at least for several passages. In species other than the mouse, ES cell technology is still under development and there are no published data reporting germ line transmission in any species other than mouse.
Recombinant Leukemia Inhibitory Factor (LIF) is presently routinely added to the culture medium used for the isolation of embryonic stem (ES) cells from mammalian embryos in vitro. This method is claimed in U.S. Pat. No. 5,166,065, EP 0380646 and WO9001541, based on a priority document AU1988 PI09644 dated Aug. 4, 1988 (51-53). Recombinant murine or human LIF protein was purified and cDNA cloned on the basis of its ability to induce differentiation of the murine monocytic cell line M1 in mature macrophages with consequent reduced clonogenicity. The (recombinant) protein and cDNA's (the murine and human variants) are claimed in a.o. U.S. Pat. No. 5,187,077 (and several continuations in part to up to U.S. Pat. No. 6,261,548 issued 17 Jul. 2001) and EP 285448, based on a priority document of AU1987 PI1209 dated Apr. 2, 1987.
Subsequent work has established the identity of LIF with earlier purified proteins and/or biological activities. The work of Hozumi et al. during 1980-1986 led to the purification to homogeneity of Factor D which stimulated the differentiation and inhibited the proliferation of the murine monocytic cell line M1, Tomida M, Yamamoto-Yamaguchi Y, Hozumi M. Purification of a factor inducing differentiation of mouse myeloid leukemic M1 cells from conditioned medium or mouse fibroblast L929 cells. J Biol Chem 1984; 259: 10978-82). The Factor D cDNA was subsequently shown to be identical to that of LIF (Lowe D G, Nunes W, Bombara M, McCabe S, Ranges G E, Henzel W, Tomida M, Yamamoto-Yamaguchi Y, Hozumi M, Goeddel D V. Genomic cloning and heterologous expression of human differentiation-stimulating factor. DNA 1989; 8: 351-9). The use of LIF in the culture medium of ES cells was preceeded by work on the inhibition of the differentiation of murine embryonic stem cells by DIA (differentiation inhibiting activity) secreted by Buffalo rat liver cells. Subsequently the identity of DIA and LIF was established at the cDNA and protein level (Smith A G, Heath J K, Donaldson D D, Wong G C, Moreau J, Stahl M, Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 1988; 336: 688-90; Smith A G, Nichols J, Robertson M, Rathjen P D. Differentiation inhibiting activity (DIA/LIF) and mouse development. Devel Biol 1992; 151: 339-51.).
Advances in recombinant DNA technology over the last decade have greatly facilitated the isolation and manipulation of genes, to the point where any conceivable novel construct can be engineered, such as by fusing the promoter of one gene to the coding sequence of another, or by site-directed mutagenesis. Likewise, advances in embryo manipulation have facilitated the transfer of these novel exogenous genes into endogenous chromosomal DNA, generating transgenic animals. Transgenic animals can be generated either by injection of DNA into the pronucleus of zygotes, by introduction of (genetically manipulated) pluripotent embryonic stem (ES) cells into host “embryos”, and more recently by nuclear transfer with stably transfected somatic donor cells into enucleated oocytes.
The review of the current technology shows that there is a need for economic compositions that provide ES cells which remain pluripotent and germ line competent after prolonged passaging. There is also a need for the generation of transgenic mice of strains with different genetic background and for the generation of other non human transgenic mammals. These transgenic animals could be useful for the study of the biological effects of identified genes, for the pharmaceutical production of therapeutic gene products, for the generation of “improved” live stock, etc.
The difficulties in maintaining the undifferentiated phenotype of cultivated stem cells is not limited to embryonic stem cells. Also adult stem cells of different lineages tend to loose their capacity to differentiate in different cell types. The maintenance of the stem cell phenotype is especially challenging for hematopoietic stem cells.
A hematopoietic stem cell (abbreviated as HSC) is a cell isolated from peripheral blood, umbilical cord blood or bone marrow that can renew itself, can differentiate to a variety of specialized cells. The bone marrow HSC can mobilize out of the bone marrow. HSC can undergo programmed cell death, called apoptosis—a process by which cells that are detrimental or unneeded self-destruct. About 1 in every 10,000 to 15,000 bone marrow cells is thought to be a stem cell. In the blood stream the proportion falls to 1 in 100,000 blood cells. (Stem cells: scientific progress and future research directions June 2001, NIH)
During the last 30 years, transplantation of hematopoietic progenitor/stem cells from bone marrow and mobilized peripheral blood is a procedure of unquestioned clinical utility and a standard of care in a number of malignancies, benign and dysplastic hematologic disorders and inherited diseases (Dupont B. (1997) in Immunology Reviews, Vol. 157, 5-12.)
Hematopoietic transplants are especially successful in treatments involving high dose chemotherapy or radiation aiming to destroy existing diseased blood cells or tumors, thereby limiting the blood, stroma and immune patients' ability to regenerate cells of the blood and immune system. The donated stem cells are infused into a patient's vein and if the transplant is successful, the donated hematopoietic stem cells will grow in number and restore the recipient's marrow and its blood-forming function.
Despite expanding roles for autologous bone marrow or peripheral blood stem cells, many indications require allogeneic transplantation, due to the potential risk of transferring malignant cells with the transplant in patients with malignant disease after autologous stem cell transplantation, and also, because the number of available human leukocyte antigen (HLA)-identical siblings is often limited. The principal limitations of allogeneic bone marrow transplantation are the lack of suitable fully HLA-matched donors and the complications of graft-versus-host disease associated with HLA-disparities.
The finding that placenta blood, also known as umbilical cord blood (UCB) contains high numbers of HSC, a comparable frequency of myeloid and erythroid progenitors to adult bone marrow, a higher proportion of immature colony-forming cells, a dec7reased risk of transmission of infection and higher percentage of telomerase, promises to circumvent many of the problems (Mayani H, Lansdorp P. in (1998) Stem Cells. 16, 153-165). The success of the first transplant performed in 1988 in a Fanconi's anemia patient (Gluckman E. et al (1989) in N. Engl. J. Med., 321: 1174-8) has proven that human UCB is a feasible alternative source of HSCs and prompted the development of large worldwide Cord Blood banking programs (Sirchia G. and Rebulla P. (1999) in Haematologica, 84: 738-747)
The most important disadvantage of umbilical cord blood is that average donations contain only 1.5 109 nucleated cells as average, one tenth of the nucleated cell (NC) dose conventionally used for bone marrow transplants in adults (Fasouliotis S, and Schenker J. (1999). Eur. J. Obst. Gynecol. Reprod. Biol. 9: 13-25). Doctors are rarely able to extract more than a few million UCB HSCs, too few to use in a transplant for an adult, who would ideally get 7 to 10 million CD34+ cells per kilogram body weight (b.w.) but often adequate for a transplant for a child even though a frequent discrepancy still exists between the number of CD34+ cells reinfused and the engraftment efficiency. Recently some authors proposed a threshold dose of 5×104 CD34+CD38− cells/kg b.w. below which the trilineage engraftment kinetics are significantly slower and unpredictable (Henon P H et al. (2001) in J. Biol. Regul. Homeost. Agents. 15, 62-67). This lower NC number implies potential limitations for the widespread use of the cord blood. Therefore a major challenge in stem cell research is the development of ex vivo culture conditions that facilitate in vitro maintenance and expansion of long term transplantable HSCs. Establishment of such culture systems is a prerequisite for potential ex vivo manipulation and expansion of transplantable HSCs in several clinical applications such as gene therapy, tumor cell purging, and stem cell transplantation.
There is a need for suitable laboratory conditions wherein stem cells can be stimulated to expand without losing their stem cell properties, thus increasing the dose of transplantable cells derived from a single donor to a transplant patient. Expansion of HSCs has proven problematic and scientists face major roadblocks in expanding their use beyond the replacement of blood and immune system. First, HSCs are unable to proliferate (replicate themselves) and differentiate (become specialized to other cell types) in vitro (Lagasse E., Weissman I L., (2001) in Immunity, 14, 425-436.). Secondly scientists do not have yet an accurate idea of the identity of a true stem cell. Although the ex vivo expansion of long term repopulating cells under stroma free conditions has not yet been achieved in a reproducible way, it is suggested that the continued quest for mechanisms that govern the proliferation and differentiation of hematopoietic stem cells could lead to the development of culture systems that expand not only committed progenitors but also HSCs (Verfaillie C. (2002). in Nature Immunol. 3, 314-317). Lewis I. et al (2001) in Blood 97, 3441-3449, present the expansion of HUC derived CD34+ cells in non-contact cultures with feeder cells with a combination of cytokines and show that these expanded cells can repopulate engrafted NOD/SCID (Non Obese Diabetic/Severe Combined Immunodeficient) mice. Piacibello et al (1997) in Blood 89, 2644-2653 show that CD34+ HUC derived cells rapidly decrease in number and die within three weeks in cultures without added cytokines. Piacibello et al (1999) in Blood 93, 11, 3736-3749 describe that CD341 HUCcells can be expanded for up to 10 weeks in stroma-free cultures in the presence of a cocktail of growth factors without losing their in vivo repopulating potential as assayed by the repopulation of sublethally irradiated NOD/SCID mice. There is thus still a need for novel or alternative media with or without added growth factors for the expansion of adult stem cells and early progenitor cells such as HSC.